1. Field of the Invention
This invention relates to a polyhydroxyalkanoate (hereinafter, referred to as a xe2x80x9cPHAxe2x80x9d) synthase, a gene encoding the synthase, a recombinant vector containing the gene, a transformant transformed by the vector, a process for producing the PHA synthase utilizing the transformant, and a process for preparing the PHA utilizing the transformant.
2. Related Background Art
There have been reported a number of microorganisms producing poly-3-hydroxybutyric acid (PHB) or another PHA and storing it therein (xe2x80x9cBiodegradable Plastic Handbookxe2x80x9d, edited by Biodegradable Plastic Research Society, NTS Co. Ltd., p. 178-197 1995). These polymers may be, as conventional plastics, used for producing a variety of products by, for example, melt processing. Since they are biodegradable, they have an advantage that they can be completely degraded by microorganisms in the natural environment, and they do not cause pollution due to remaining in the natural environment like many conventional polymer compounds. Furthermore, they are excellently biocompatible, and thus are expected to be used in applications such as a medical soft member.
It is known that a composition and a structure of such a PHA produced by a microorganism may considerably vary depending on the type of a microorganism used for the production, a culture-medium composition and culturing conditions. Investigations have been, therefore, mainly focused on controlling such a composition or structure for the purpose of improving physical properties of a PHA.
For example, Japanese Patent Application Nos. 7-14352 and 8-19227 and Japanese Examined Publication No. 6-15604 describe that Alcaligenes eutropus H16 (ATCC No. 17699) and its variants may produce 3-hydroxybutyric acid (3HB) and its copolymer with 3-hydroxyvaleric acid (3HV) with various composition ratios by changing a carbon source during culturing.
Japanese Patent Publication No. 2642937 has disclosed that PHA in which a monomer unit is 3-hydroxyalkanoate with 6 to 12 carbon atoms may be produced by supplying a non-cyclic aliphatic hydrocarbon as a carbon source to Pseudomonas oleovorans (ATCC No. 29347).
Japanese Patent Application Laid-Open No. 5-7492 discloses methods in which Methylobacterium sp., Paracoccus sp., Alcaligenes sp., and Pseudomonas sp. are contacted with a primary alcohol with 3 to 7 carbon atoms to produce a copolymer of 3HB and 3HV.
Japanese Patent Application Laid-Open No. 5-93049 and No. 7-265065 have disclosed that Aeromonas caviae is cultured using oleic acid or olive oil as a carbon source to produce a two-component copolymer of 3HB and 3-hydroxyhexanoic acid (3HHx).
Japanese Patent Application Laid-Open No. 9-191893 has disclosed that Comamonas acidovorans IF013852 is cultured using gluconic acid and 1,4-butanediol as carbon sources to produce a polyester having 3HB and 4-hydroxybutyric acid as monomer units.
Furthermore, it has been reported that certain microorganisms produce PHAs having a variety of substituents such as groups derived from an unsaturated hydrocarbon, ester, allyl, cyano, groups derived from a halogenated hydrocarbon and epoxide. Recently, there have been attempts for improving physical properties of a PHA produced by a microorganism using such a procedure.
As an example of such a polymer, a PHA having a phenyl group in its side chain has been developed. For example, Makromol Chem., 191, 1957-1965 (1990); Macromolecules, 24, 5256-5260 (1991); and Chirality, 3, 492-494 (1991) have described production of a PHA comprising 3-hydroxy-5-phenylvaleric acid (3HPV) as a monomer unit by Pseudomonas oleovorans, where there has been observed variation in polymer physical properties probably due to the presence of 3HPV.
As described above, microorganism-produced PHAs with various combinations of composition and structure have been obtained by varying factors such as the type of a microorganism used, a culture medium composition and culturing conditions. However, each microorganism or PHA synthase has significantly different substrate specificity. Therefore, it has been difficult to produce PHAs comprising different monomer units extensively suitable to a variety of applications using known microorganisms or PHA synthases alone.
A PHA having a substituent in its side chain as described above may be expected to be a xe2x80x9cfunctional polymerxe2x80x9d having significantly useful functions and properties owing to the properties of the introduced substituent. It is, therefore, extremely useful and important to prepare a gene encoding a PHA synthase from a microorganism which can produce and store a very useful polymer having both such functionality and biodegradability; prepare a recombinant vector comprising the gene, a transformant transformed by the vector; and develop a process for producing a PHA synthase utilizing the transformant and a process for preparing a PHA utilizing the transformant.
In view of usefulness of such a PHA synthase useful in PHA production, an object of the present invention is to provide a PHA synthase, a gene encoding the enzyme, a recombinant vector comprising the gene, a transformant transformed by the vector, a process for producing a PHA synthase utilizing the transformant and a process for preparing a PHA utilizing the transformant.
For developing a PHA having a novel side-chain structure useful as, for example, a device material or a medical material, the inventors have searched a novel microorganism capable of producing and storing the desired PHA. Additionally, the inventors have intensely investigated for preparing a gene encoding a PHA synthase from such a microorganism, a recombinant vector containing the gene, a transformant transformed by the vector and developing a process for producing a PHA synthase utilizing the transformant and a process for preparing a PHA utilizing the transformant.
The inventors have finally found a novel microorganism capable of producing and storing a novel PHA comprising 3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) represented by formula (2) as a monomer unit from synthetic 5-(4-fluorophenyl)valeric acid (PPVA) represented by formula (1) as a starting material, and designate it as P91 strain. 
The inventors have also found that P91 strain in capable of producing and storing a PHA comprising 3-hydroxy-4-phenoxy-n-butyric acid (3HPxB) represented by formula (4) as a monomer unit from 4-phenoxy-n-butyric acid (PxBA) represented by formula (3) as a starting material. 
An example of a microorganism capable of producing and storing a PHA comprising 3HPxB as a monomer unit is Pseudomonas oleovorans involved in a process described in Macromolecules. 29, 3432-3435, 1996. This process is considerably different from the process using PxBA as a substrate in P91 strain in that 8-phenoxyoctanoic acid (PxOA) is used as a substrate. In addition, for a PHA produced, the above reported process provides a copolymer consisting of three monomer units, i.e., 3-hydroxy-8-phenoxyoctanoic acid derived from the substrate PxOA, 3-hydroxy-6-phenoxyhexanoic acid as a byproduct derived from a metabolite and 3HPxB. On the other hand, P91 strain can produce a PHA comprising 3HPxB derived from PxBA as a sole phenoxy-containing monomer unit. In this respect, P91 strain is basically different from the above reported strain.
There are no reports describing microbial production of a PHA comprising 3HPxD as a monomer unit using PxBA as a substrate or 3HPxB as a sole phenoxy-containing monomer unit.
Microbiological properties of P91 strain according to this invention are as follows.
 less than Microbiological Properties of P91 Strain greater than 
(Morphologic Properties)
Cell shape and size: Bacilliform, 0.6 xcexcmxc3x971.5 xcexcm
Cell polymorphism: No
Motility: Yes
Sporulation: No
Gram stainability: Negative
Colonization: Circular, smooth in the overallperiphery, low convex, smooth surface, gloss, cream color
(Physiological Properties)
Catalase: Positive
Oxidase: Positive
O/F test: oxidized form
Reduction of a nitrate: Negative
Indole formation: Negative
Acidification of dextrose: Negative
Arginine dihydrolase: Positive
Urease: Negative
Esculin hydrolysis: Negative
Gelatin hydrolysis: Negative
xcex2-Galactosidase: Negative
Fluorochrome production on King""s B agar: Positive
(Substrate Assimilation Ability)
Dextrose: Positive
L-Arabinose: Negative
D-Mannose: Negative
D-Mannitol: Negative
N-Acetyl-D-glucosamine: Negative
Maltose: Negative
Potassium gluconate: Positive
n-Capric acid: Positive
Adipic acid: Negative
dl-Malic acid: Positive
Sodium citrate: Positive
Phenyl acetate: Positive
From these microbiological properties, the inventors have attempted to categorize P91 strain according to Bergey""s Manual of Systematic Bacteriology, Volume 1 (1984) and Bergey""s Manual of Determinative Bacteriology 9th ed. (1994) to determine that the strain belongs to Pseudomonas putida. Thus, the strain was designated as Pseudomonas putida P91. The inventors have deposited Pseudomonas putida P91 to Patent Microorganism Depository Center in the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, under the deposition number of FERM P-17409. P91 strain has been internationally deposited on the basis of the Budapest Treaty, and its international deposition number is xe2x80x9cFERM BP-7373xe2x80x9d.
The inventors have intensely conducted investigation for solving the above problems and finally have succeeded cloning a gene for a PHA synthase from P91 strain to achieve this invention.
Specifically, a PHA synthase of this invention is characterized in that it has the amino acid sequence of SEQ ID NO.:1 or 3. A PHA synthase according to the present invention may include a mutant PHA synthase where at least one mutation including deletion, substitution or addition of at least one amino acid is introduced as long as it does not deteriorate PHA synthase activity exhibited by a protein comprising these amino acid sequences.
The present invention also encompasses a PHA synthase gene coding a PHA synthase comprising the amino acid sequence of SEQ ID NO.:1 or 3. Examples of a sequence of such a gene include SEQ ID NO.:2 or 4. Furthermore, a mutant PHA synthase gene encoding the above mutant PHA synthase obtained by mutation of the sequence of SEQ ID NOs.:2 and 4 is included in a PHA synthase gene according to this invention.
The present invention also encompasses a recombinant vector comprising the above PHA synthase gene and a transformant transformed by the recombinant vector. The present invention also encompasses a process for producing a PHA synthase comprising the steps of culturing the transformant and isolating the PHA synthase from a culture obtained, and a process for preparing a PHA comprising the steps of culturing the transformant and isolating the PHA from a culture obtained.
The present invention provides a PHA synthase, a gene encoding the PHA synthase, a recombinant vector comprising the gene and a transformant transformed by the recombinant vector. The PHA synthase gene according to the present invention is useful for preparing a PHA having various physical properties because it encodes a PHA synthase using a monomer having a novel side-chain structure as a substrate.